JP2006514698A - Nanoparticle bioactive substances - Google Patents

Abstract

The bioactive substance is reproducibly converted into particles having a diameter of about 5 to about 2000 nanometers (nm). The conversion involves dissolving the bioactive substance in a solvent for the bioactive substance; for example, diluting the bioactive substance solution using an excess liquid that is non-solvent for the bioactive substance but miscible with the solvent. By quickly changing the polarity of the solution to make it a non-solvent for the bioactive substance. The precipitated bioactive agent nanoparticles are collected by centrifugation, filtration or lyophilization. The nanoparticles have a relatively narrow particle size distribution and the average particle size can be adjusted by the choice of solvent and non-solvent. Nanoparticles are generally amorphous. A surfactant can be added to ensure dispersion of the particles upon administration. In a preferred embodiment, the physiologically active substance is a drug having low water solubility.

Description

  The present invention relates to compositions containing biologically active substances in nanoparticulate form with increased dissolution and / or uptake rates.

  This application is filed in U.S. Patent Application No. 60 / 4,230,93 filed on October 30, 2002, and U.S. Patent Application No. 60 / 4,903,43 filed on July 25, 2003. Claim priority.

(Background of the Invention)
Paclitaxel is a drug with extremely low water solubility and exhibits extremely low GI absorption after oral administration. Common methods of parenteral delivery of poorly soluble drugs include the use of large amounts of aqueous diluents, solubilizers, detergents, non-aqueous solvents, or physiological pH solutions. However, these formulations can increase the systemic toxicity of the pharmaceutical composition or damage body tissue at the site of administration.

  For example, paclitaxel is a natural product that has been shown to have cytotoxic and antitumor activity. Although excellent therapeutic potential is well known, paclitaxel has several drawbacks related to patients as a therapeutic agent. These are due in part to extremely low water solubility making it difficult to provide in a suitable dosage form. Due to the low water solubility of paclitaxel, the currently approved (US FDA) clinical formulation is a solution of 6 mg / mL paclitaxel in 50% hydroxyethylated castor oil (CREMOPHOR EL®) and 50% absolute alcohol. Consists of. Am. J. et al. Hosp. Pharm, 48: 1520-24 (1991). In some cases, severe reactions, including hypersensitivity, occur in association with CREMOPHOR® administered with paclitaxel to compensate for the low water solubility of paclitaxel. Due to the occurrence of hypersensitivity reactions to commercial paclitaxel formulations and the possibility of paclitaxel sedimentation in the blood, the formulation must be infused over several hours. In addition, patients must be pretreated with steroids and antihistamines prior to infusion.

  Other efforts to increase the dissolution rate focus on transporting the drug as a dispersion in a water soluble or biodegradable matrix, generally in the form of polymer microparticles. For example, it has been reported that the dissolution rate of dexamethasone was improved by entrapment of the drug in chitosan microspheres formed by spray drying (Genta et al., ST P. Pharma Sciences 5 (3): 202-. 07 (1995)). Similarly, an increased dissolution rate has been reported by mixing water-soluble gelatin, which is said to make the drug surface hydrophilic, and a low-solubility drug powder (Imai et al., J. Pharm. Pharmacol., 42: 615). -19 (1990)). Related efforts are directed to forming relatively large porous matrices of poorly soluble drugs. For example, Roland & Paerakul, “Spherical Aggregates of Water-Insoluble Drugs”, J. Am. Pharma. Sci. 78 (11): 964-67 (1989) discloses the production of beads having a low soluble drug content of up to 98%, which have a porous internal structure.

  In particular, taxol formulations which are said to have improved transport (including oral transport) properties are described in WO 01/30319, WO 01/57013, WO 01/30448, US Pat. No. 6,334,445. No. 6,245,805, WO 98/53811, WO 97/15269, WO 00/78247 and US Pat. No. 5,969,972. All of these are limited in terms of actual bioavailability. For an orally administered taxane, a bioavailability of at least 10% is considered essential for commercial success, but even for formulations that give lower levels of oral availability, especially for cancer It may still have use in prophylactic therapy for individuals at risk or receiving cancer treatment.

  One factor that affects drug dosage is the rate of dissolution of the drug in body fluids. Adjustment of the dissolution rate is considered important to obtain the desired therapeutic effect. If the drug is readily soluble, its dissolution rate can be reduced by various modified release methods. In general, dissolution control methods are performed by coating the drug particles with a dissolution-retarding coating, or by slowly dissolving or forming a disintegrating tablet. Coatings or tableting materials (eg polymers) may dissolve slowly, or they may actually be insoluble, by in situ degradation of the coating or polymer, or of the drug from the coating or polymer. The drug may be released by diffusion.

  Control of dissolution rate is more difficult when the drug is poorly soluble in body fluids. Not easily dissolving can mean that the drug is less bioavailable, especially in oral dosage forms where there is a limited transit time through the gastrointestinal (GI) tract. In the case of such drugs, the goal is to find a way to transport them to the tissue faster than their inherent insolubility is acceptable. One method is to dissolve them in a non-aqueous solvent. An alcoholic extract or solution of the drug can be formed. Recently, hydrophobic drugs, such as paclitaxel, are dissolved in castor lipid, the solution is emulsified and then intravenously injected as an emulsion. For example, US Pat. No. 6,334,445 to Mettinger discloses such a procedure. This procedure has side effects including allergic reactions to paclitaxel, reversible myelosuppression, myalgia, mucositis and alopecia. Therefore, there is a need for improvement.

  US Pat. No. 6,143,211 to Mathiowitz et al. And US Pat. No. 6,368,586 to Jacob et al. Disclose methods of transporting drugs into the circulation through the intestine using coated drug particles. ing. In addition to slow release in the intestine, some of the improved transport is believed to be due to particles induced to pass between or through cells on the mucosal surface (Mathiowitz et al., Nature 386: 410 (1997). )reference). In addition to the protective effect of the polymer coating, it is believed that this method more effectively incorporates smaller particle sizes.

  However, current methods of preparing drugs as small particles generally result in relatively large particles having a diameter of tens of microns to a few millimeters. Generally, the drug is manufactured by precipitation, crystallization or lyophilization or other drying methods. The resulting product is generally visible to the naked eye. In standard tableting, the size range of the drug is often not critical. The drug can be ground, ground, or otherwise subdivided to obtain a reasonably homogeneous powder that can be further processed, but the millimeter range particles are often small enough.

  Recently, efforts have been made to produce sub-millimeter particles, generally for use in controlled release formulations. These methods most commonly involve grinding or attrition, but other methods are also known.

  Mathiowitz et al., US Pat. No. 6,235,224, and Mathiowitz et al., Nature 386: 410 (1997) disclose methods for encapsulating drugs in micron and submicron polymer microspheres. In this method, referred to as “phase inversion nanoencapsulation” (“PIN”), the polymer is dissolved in a solvent and the drug or other substance to be encapsulated is dissolved or suspended in the polymer solution. The resulting solution or suspension is quickly diluted using a solution that is non-solvent for the polymer and preferably the drug or substance. The non-solvent is selected to be sufficiently miscible with the solvent, thereby forming a single phase solution that is non-solvent for the polymer after dilution. Spontaneous mixing of the two solutions occurs quickly and with a small characteristic scale of mixing. As a result, the polymer precipitates to form very small diameter particles, typically up to tens to hundreds of nanometers, or in some cases up to several microns. These particles are approximately uniform in size. A drug or substance is encapsulated in the nanosphere. Upon administration to a patient or other application, drug or substance is released from the nanosphere by polymer diffusion, degradation, or a combination of these effects.

  However, in some situations, the presence of encapsulated polymer may be unnecessary or even inhibit drug transport. For example, in the transport of highly hydrophobic or otherwise poorly soluble drugs where drug dissolution is rate limiting in transport, to slow the drug transport or to protect the drug from the effects of transport pathways in the body Does not require a coating. Examples of such transport routes include the circulatory system, gastrointestinal tract, urinary and genital tract, mucosa and skin.

  Accordingly, it is an object of the present invention to provide a composition for rapid delivery of drugs.

  Another object of the present invention is to provide a method of forming drug particles that increases drug transport rate and effectiveness.

  Another object of the present invention is to provide an orally administrable formulation that provides clinically acceptable levels of taxanes.

(Summary of the Invention)
Biologically active substances can be reproducibly converted to particles having a diameter of about 5 to about 2000 nanometers (nm). Conversion involves quickly changing the polarity of a solution by dissolving the substance in a solvent for the substance and diluting the substance solution using an excess of liquid that is non-solvent for the substance but miscible with the solvent, for example. The solvent is made non-solvent with respect to the substance particles. The precipitated material nanoparticles are collected by centrifugation, filtration or lyophilization. The nanoparticles have a relatively narrow particle size distribution and the average diameter can be adjusted by the choice of solvent and non-solvent. Nanoparticles are generally amorphous. A surfactant may be added to ensure dispersion of the particles upon administration. In a preferred embodiment, the substance is a drug. In the most preferred embodiment, the substance is a drug with low water solubility.

(Detailed description of the invention)
(I. Nanoparticle composition)
The particles are for the most part a population of nanoparticles generally greater than 95%, more preferably greater than 99%, less than 1 micron in diameter, generally stable and not irreversibly aggregated.

  The prepared particles have a volume average diameter of less than about 1 micron. They are generally less than about 5 microns in diameter (number average or volume average diameter), more typically less than 1 micron, often from about 500 nm to about 50 nm, although smaller average diameters are also obtained. Particle dispersion is relatively narrow and generally not monodisperse.

  FIG. 1 shows the particle size results for a typical drug nanoparticle preparation. The number average diameter is reasonably symmetric and is concentrated at about 80 nm. The volume average diameter peaks at about 200 nm and there are virtually no particles larger than about 700 nm.

  In contrast to conventional drug particles, which are generally crystalline, the nanoparticles are generally amorphous in structure. That it is not crystalline can be observed by microscopy or by methods such as differential scanning calorimetry (DSC). If the drug in question is poorly soluble in bodily fluids, non-crystalline particles will have an increased drug dissolution rate compared to crystalline particles of the same drug.

  Typically, nanoparticles consist essentially of drugs. As used herein, “drug” refers to any biologically active substance, including standard small molecule drugs, therapeutically effective proteins, lipids, polysaccharides, proteoglycans and polynucleotides. It is not limited to them. The agent may be a therapeutic, prophylactic or diagnostic agent.

  Any agent that can be converted to a microparticle or nanoparticle material can be formed into nanoparticles using the methods described herein. The drug may be in either small molecule or macromolecular form. The drug may be poorly soluble in body fluids or soluble in body fluids. For example, very small particle sizes are useful for delivery as aerosols to the nasal passages and nasal passages or lungs. This method is also useful for the production of dosage forms of shear sensitive agents such as proteins and nucleic acids. Many drugs are known and listed in standard handbooks such as Merck Index and Physicians Desk Reference.

  In the most preferred embodiment, the drug is poorly soluble in body fluids. For example, the drug may be less than about 0.1% w / v water soluble at room temperature. Their low solubility is thought to be due to the slow dissolution rate or inherent low solubility. These agents are often hydrophobic, such as bioavailability class II and IV agents. Many therapeutically important drugs with low solubility are known. Examples include taxanes such as paclitaxel and docetaxel; camptothecin; cyclosporine and related immune response inhibitors; griseofulvin, itraconazole and related antifungal drugs; metromidazole and related antidysenteric drugs; dicoumarol and related anticoagulants; Steroids such as androsterone and estradiol. Agents having low water solubility, such as agents having water solubility similar to those described above, can also be used. Taxanes are anticancer cytotoxins that stabilize cellular microtubules. Taxane compounds useful in the compositions and methods disclosed herein include paclitaxel and docetaxel, and their natural and synthetic analogs that have anticancer or antiangiogenic activity. Paclitaxel and docetaxel have considerable activity, and one or both of these drugs are widely recognized as therapeutic ingredients for advanced breast, lung and ovarian cancer.

  The formulation may contain a taxane with a drug loading of up to 70 wt%. In a preferred embodiment, the taxane is present at a drug loading of 30-70 wt%. The taxane may be present at a drug loading of about 30-50 wt%. The formulation may contain low levels of drug loading, eg, about 10-30 wt% taxane, or about 1-10 wt% taxane.

(B. Excipients and carriers)
The nanoparticles may be used alone or coated with one or more surfactants (“surfactants”), polymers, adhesion promoters, or other additives or excipients. The nanoparticles may be incorporated or encapsulated in tablets or capsules or other dosage forms. A variety of excipients are commonly used in pharmaceutical formulations. Excipient types include, but are not limited to, tableting aids, disintegrants, glidants, antioxidants or other preservatives, enteric coatings, flavoring agents, and the like. Literature describing such substances is readily available and well known to those skilled in the pharmaceutical formulation arts. The excipient may be added at any of the stages described below in which the particles contain a surfactant. For example, excipients can be added during microparticle formation; while the microparticles are dispersed to form the dosage form; or during administration of the microparticles.

  The choice of additive or excipient is determined in part by the intended route of administration. Any common route (eg, inhalation, oral, rectal, vaginal, topical and parenteral) is suitable for and can be improved by the use of nanoparticulate drug formulations. Suitable formulations include oral formulations, aerosols, topical formulations, oral formulations, and implantable compositions. In particular, nanoparticulate drug formulations are suitable for delivering hydrophobic and other poorly soluble drugs, such as bioavailability class II and IV drugs, by oral or aerosol administration, thereby providing a parenteral route of administration. Can replace it.

(Surfactant)
The nanoparticles can optionally contain a surfactant to remove or reduce particle agglomeration. The surfactant adheres to the surface of the nanoparticles. In general, a surfactant is either an initial non-solvent mixture in which particles are formed, a medium in which nanoparticles are taken up for administration, and a medium in which the particles are subsequently transported (eg, gastrointestinal fluid). Promotes dispersion of nanoparticles in one or all.

  Any surface activity can be used for the nanoparticles. Suitable surfactants include both small molecule surfactants, often referred to as detergents, and macromolecules (ie polymers). The surfactant may contain a surfactant mixture. In formulations for parenteral administration, the surfactant is preferably a surfactant approved for pharmaceutical use by the FDA. In formulations for administration that is not parenteral, the surfactant may be a surfactant approved by the FDA for use in food or cosmetics.

  The surfactant may be present in any suitable amount. In a preferred embodiment, the effective surfactant is present only as a low weight part of the nanoparticles, eg, 0.1% to 10% (surfactant weight / drug weight). However, especially when the particles are small and the total surface area is accordingly large, a larger proportion of surfactant may be required or convenient, so the surfactant is 20 wt% of the drug weight, It may be present at 50 wt% or up to about 100 wt%.

  The choice of surfactant is necessarily somewhat empirical and some surfactants may be ineffective in certain applications. For example, the following example shows that 0.5% sodium lauryl sulfate (SLS), a surfactant that is effective in many applications, is not effective in dispersing paclitaxel microparticles as they pass through the gastrointestinal tract. .

(polymer)
Suitable polymers include hydrogels, thermoplastic resins, and soluble and water-insoluble and biodegradable and non-biodegradable polymers including homopolymers, copolymers and blends of natural and synthetic polymers. Typical polymers that can be used include hydrophilic polymers such as carboxyl group-containing polymers including polyacrylic acid. Bioerodible polymers can also be used, including polyanhydrides, poly (hydroxy acids) and polyesters and blends and copolymers thereof. Typical bioerodible poly (hydroxy acids) and copolymers thereof that can be used are poly (lactic acid), poly (glycolic acid), poly (hydroxy-butyric acid), poly (hydroxyvaleric acid), poly (caprolactone), poly (Lactide-co-caprolactone) and poly (lactide-co-glycolide). Polymers containing labile bonds, such as polyanhydrides and polyorthoesters, can be used in any modified form with reduced hydrolysis reactivity. Positively charged hydrogels such as chitosan, and thermoplastic polymers such as polystyrene can also be used.

  Exemplary natural polymers that can be used include proteins such as zein, modified zein, casein, gelatin, gluten, serum albumin, or collagen, and polysaccharides such as dextran, polyhyaluronic acid, and alginic acid. Exemplary synthetic polymers include polyphosphazenes, polyamides, polycarbonates, polyacrylamides, polysiloxanes, polyurethanes and copolymers thereof. Cellulose can also be used. “Cellulose” as defined herein includes natural and synthetic celluloses such as alkyl celluloses, cellulose ethers, cellulose esters, hydroxyalkyl celluloses and nitrocelluloses. Examples of cellulose include ethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate lactate, cellulose acetate phthalate, cellulose triacetate, and An example is cellulose sulfate sodium salt.

  Polymers of acrylic and methacrylic acid or esters, and copolymers thereof can be used. Typical polymers that can be used include poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl). Methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), and poly (octadecyl acrylate).

  Other polymers that may be used include polyalkylenes such as polyethylene and polypropylene; polyarylalkylenes such as polystyrene; poly (alkylene glycols) such as poly (ethylene glycol); poly (alkylene oxides) such as poly (ethylene oxide); and , Poly (alkylene terephthalate), such as poly (ethylene terephthalate). In addition, polyvinyl polymers can also be used, and the polyvinyl polymers defined herein include polyvinyl alcohol, polyvinyl ether, polyvinyl ester and halogenated polyvinyl. Examples of polyvinyl polymers include poly (vinyl acetate), polyvinylphenol and polyvinylpyrrolidone.

  Polymers that change viscosity as a function of temperature or shear or other physical forces may also be used. Poly (oxyalkylene) polymers and copolymers, such as poly (ethylene oxide) -poly (propylene oxide) (PEO-PPO) or poly (ethylene oxide) -poly (butylene oxide) (PEO-PBO) copolymers, and these polymers; Copolymers and blends with polymers such as poly (α-hydroxy acids) (including but not limited to lactic acid, glycolic acid, hydroxybutyric acid, polycaprolactone and polyvalerolactone) can be synthesized or obtained commercially Yes. For example, polyoxyalkylene copolymers are described in US Pat. Nos. 3,829,506; 3,535,307; 3,036,118; 2,979,578; 2,677,700; And 2,675,619.

  These polymers are available from Sigma Chemical Co. , St. Polysciences, Warrenton, PA; Aldrich, Milwaukee, WI; Fluka, Ronkonoma, NY; and sources such as Biorad, Richmond, CA, or other sources using standard methods It can be synthesized from monomers obtained from the company.

  The polymers are selected based on their bioadhesive properties, for example as disclosed in Mathiowitz et al. US Pat. Nos. 6,197,346; 6,217,908; and 6,235,313. can do.

  Polymers that can be used include both non-biodegradable or biodegradable, synthetic and natural polymers. Exemplary synthetic polymers include polyethylene glycol (“PEG”), polyvinyl pyrrolidone, polymethacrylate, polylysine, poloxamer, polyvinyl alcohol, polyacrylic acid, polyethylene oxide, and polyethyazoline. Typical natural polymers include albumin, alginate, gelatin, gum arabic, chitosan, cellulose dextran, ficoll, starch, hydroxyethylcellulose, hydroxypropylcellulose, hydroxy-propylmethylcellulose, hyaluronic acid, carboxyethylcellulose, carboxymethylcellulose, deacetylated Chitosan, dextran sulfate and their derivatives. Preferred hydrophilic polymers include PEG, polyvinyl pyrrolidone, poloxamer, hydroxypropyl cellulose and hydroxyethyl cellulose. The hydrophilic polymer is selected for use based on various factors such as polymer molecular weight, polymer hydrophilicity, and polymer intrinsic viscosity.

(Wetting agent)
Representative examples of wetting agents include mannitol, dextrose, maltose, lactose, sucrose, gelatin, casein, lecithin (phosphatide), gum arabic, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate Rate, cetostearyl alcohol, cetomacrogol emulsified wax, sorbitan ester, polyoxyethylene alkyl ether (eg, macrogol ether such as cetomacrogol 1000), polyoxyethylene castor oil derivative, polyoxyethylene sorbitan fatty acid Esters (eg TWEEN®), polyethylene glycol, polyoxyethylene stearate, colloidal silicon dioxide, phosphine Sodium dodecyl sulfate, carboxymethylcellulose calcium, sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol and polyvinylpyrrolidone (PVP) Is mentioned.

  Tyloxapol (a nonionic liquid polymer of the alkyl aryl polyether alcohol type; also known as superinone or triton) is also a useful wetting agent. The majority of these wetting agents are known pharmaceutical excipients and are described in detail by the American Pharmaceutical Association and the Pharmaceutical Society of Great Pest, Physic, published by The Pharmaceuticals of the Pharmaceutical Company, The Pent.

Preferred dispersants include: polyvinyl pyrrolidone, polyethylene glycol, tyloxapol, poloxamers such as PLURONIC® F68, F127 and F108 (block copolymers of ethylene oxide and propylene oxide), and polyxamines such as TETRONIC ( ® 908 (also known as POLOXAMINE ® 908) (tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine) (available from BASF), dextran, lecithin, sodium sulfo Dialkyl esters of succinic acid, such as AEROSOL® OT (dioctyl ester of sodium sulfosuccinic acid) (America (Available from Cyanimid), DUPONOL® P (sodium lauryl sulfate) (available from DuPont), TRITON® X-200 (alkyl aryl polyether sulfonate) (available from Rohm and Haas) , TWEEN® 20 and TWEEN® 80 (polyoxyethylene sorbitan fatty acid ester) (available from ICI Specialty Chemicals), Carbowax 3550 and 934 (polyethylene glycol) (available from Unior Carbide), Crodesta F-110 (a mixture of sucrose stearate and sucrose distearate), and Crodesta SL-40 (both Croda In c. companies available from), and _{SA90HCO (C 18 H 37 CH 2} (CON (CH 3) CH 2 (CHOH) 4 CH 2 OH) 2).

  Wetting agents that have been found to be particularly useful include Tetronic 908, Tweens, Pluronic F-68 and polyvinylpyrrolidone. Other useful wetting agents include: decanoyl-N-methylglucamide; n-decyl-β-D-glucopyranoside; n-decyl-β-D-maltopyranoside; n-dodecyl-β- D-glucopyranoside; n-dodecyl β-D-maltoside; heptanoyl-N-methylglucamide; n-heptyl-β-D-glucopyranoside; n-heptyl-β-D-thioglucoside; n-hexyl-β-D- Nonanoyl-N-methylglucamide; n-nonyl-β-D-glucopyranoside; octanoyl-N-methylglucamide; n-octyl-β-D-glucopyranoside; and octyl-β-D-thioglucopyranoside. Another preferred wetting agent is p-isononylphenoxypoly (glycidol), also known as Olin-10G or Surfactant 10-G (commercially available as 10G from Olin Chemicals). Two or more wetting agents can be used in combination.

(Bioadhesive excipient)
Adhesion promoters are disclosed in Santos et al. US Pat. No. 6,156,348; Mathiowitz et al. US Pat. No. 6,197,346; Mathiowitz et al. US Pat. No. 6,217,908; No. 6,235,313. In some preferred embodiments, the adhesion promoter contains a hydrophobic polymer that is less hydrophobic than the drug, metal and / or metal oxide.

  The bioadhesive properties of the polymer are enhanced by incorporating metal compounds into the polymer to enhance the polymer's ability to adhere to tissue surfaces such as mucosa. Metal compounds that enhance the bioadhesive properties of the polymer are preferably water-insoluble metal compounds, such as water-insoluble metal oxides and hydroxides, including calcium, iron, copper and zinc oxides. Metal compounds can be incorporated into a variety of hydrophilic and hydrophobic polymers including proteins, polysaccharides and synthetic biocompatible polymers. In one embodiment, the metal oxide can be incorporated into a polymer used to form or coat a drug delivery device such as a microsphere containing a drug or diagnostic agent. The metal compound can be applied in the form of a fine dispersion of particles on the surface of the polymer that coats or forms the device, which enhances the ability of the device to bind to the mucosa. For example, a polymer in the form of a microsphere has an improved ability to adhere to the mucosa, and thus, through any of a variety of mucosal surfaces including the surfaces of the gastrointestinal tract, airways, drainage tract and genital tract, drugs or diagnostics Can be used to transport drugs.

  Metal compounds that can be incorporated into the polymer to improve the bioadhesive properties of the polymer include water-insoluble metal compounds such as water-soluble metal oxides and metal hydroxides, which are incorporated into the polymer and Associate and thereby improve the bioadhesiveness of the polymer. A water-insoluble metal compound as defined herein is defined as a metal compound that has little or no solubility in water, for example, a solubility of about 0.0 to 0.9 mg / mL. .

  Water insoluble metal compounds, such as metal oxides, can be incorporated by one of the following mechanisms: (a) a physical mixture that results in the capture of the metal compound; (b) ionic interaction between the metal compound and the polymer; c) surface modification of the polymer resulting in exposed metal compounds on the surface; and (d) coating methods such as flow beads, pan coating, or any similar method known to those skilled in the art to produce a metal compound rich layer on the surface of the device. Preferred properties defining the metal compound include the following properties: (a) substantially insoluble in an aqueous environment, such as an acidic or basic aqueous environment (eg, an environment present in the gastric lumen); and (b) an aqueous environment. Ionizable surface charge at pH of

  Water insoluble metal compounds can be derived from metals including calcium, iron, copper, zinc, cadmium, zirconium and titanium. For example, various water-insoluble metal oxide powders such as ferric oxide, zinc oxide, titanium oxide, copper oxide, barium hydroxide, stannic oxide, aluminum oxide, nickel oxide, zirconium oxide and cadmium oxide are used. Thus, the bioadhesive properties of the polymer can be improved. Incorporation of water-insoluble metal compounds such as ferric oxide, copper oxide and zinc oxide can significantly improve the adhesion of polymers to tissue surfaces such as mucosa, for example in the gastrointestinal system.

  In one embodiment, the metal compound is applied as a fine particle dispersion of a water-insoluble metal oxide that is incorporated into, or at least on the surface of, the polymer attached to the tissue surface. For example, in one embodiment, water-insoluble metal oxide particles are incorporated into a polymer that defines or coats microspheres or microcapsules used for drug delivery. In a preferred embodiment, the metal oxide is present on the surface of the microparticle as a fine particle dispersion. The metal compound can also be incorporated into the inner layer of the polymer device and exposed only after decomposition or dissolution of the “protect” outer layer. For example, the drug and metal containing core particles may be coated with an enteric coating designed to dissolve when exposed to gastric juice. The metal compound rich core is then exposed and can be used for binding to the GI mucosa.

  The fine metal oxide particles can be produced, for example, by pulverizing a metal oxide to form particles having a size of 10.0 to 300 nm, for example. The metal oxide particles can be incorporated into the polymer, for example, as follows: Prior to microcapsule formation, the particles are dissolved or dispersed in a polymer solution or dispersion, and then: A microcapsule formation procedure, such as one of the procedures described in detail, can be used to incorporate the polymer during macrocapsule formation. Incorporation of metal oxide particles on the surface of the microspheres advantageously enhances the ability of the microspheres to bind to the mucosa or other tissue surface and improves the drug transport properties of the microspheres.

  The metal compound incorporated into the polymer to improve the bioadhesive properties of the polymer may be a metal compound already approved by the FDA as a food or pharmaceutical additive, such as zinc oxide.

  The bioadhesive properties of the particles can also be enhanced as disclosed in US Pat. No. 6,156,348 (methods and compositions for enhancing the bioadhesive properties of polymers using organic excipients). it can. Oligomeric excipients can be blended or incorporated into various hydrophilic and hydrophobic polymers, including proteins, polysaccharides and synthetic biocompatible polymers. Anhydrous oligomers can be combined with metal oxide particles to improve bioadhesion considerably more than with organic additives alone. Incorporation of oligomeric compounds into various polymers that are generally not bioadhesive dramatically increases the adhesion of this polymer to tissue surfaces such as mucosa.

  As used herein, the term “anhydride oligomer” means a diacid or polyacid attached by an anhydride linkage and having a carboxy end group attached by a anhydride linkage to a monoacid such as acetic acid. . Anhydride oligomers are defined as having a molecular weight of less than about 5000, generally from about 100 to 5000 daltons, or having from 1 to about 20 diacid units joined by anhydride linkages. Anhydride oligomeric compounds have high chemical reactivity.

  Oligomers can be produced in the reflux reaction of diacid and excess acetic anhydride. Excess acetic anhydride is evaporated under vacuum and the resulting oligomer, which is a mixture of species containing about 1-20 diacid units linked by anhydride linkages, is crystallized from, for example, toluene or other organic solvent To be purified. The oligomer is collected by filtration, washed with, for example, ether, and the reaction yields mono- and polyacid anhydride oligomers with terminal carboxylic acid groups attached to each other by anhydride linkages.

Anhydride oligomers are hydrolytically unstable. When analyzed by gel permeation chromatography, the molecular weight is, for example, on the order of 200-400 for fumaric anhydride oligomers and 2000-4000 for sebacic acid oligomers. Anhydride bonds is generally by a characteristic double peak at 1750 cm ^-1 and 1820 cm ^-1 with a corresponding disappearance of the carboxylic acid peak at 1700 cm ^-1, can be detected by Fourier transform infrared spectroscopy.

  In one embodiment, the oligomers are, for example, Domb et al., US Pat. No. 4,757,128, Domb et al., US Pat. No. 4,997,904, and Domb et al., US Pat. The disclosure may be generated from the diacids disclosed in (herein incorporated by reference). For example, monomers such as sebacic acid, bis (p-carboxy-phenoxy) propane, isophthalic acid, fumaric acid, maleic acid, adipic acid or dodecanedioic acid may be used.

  Because of their charge and hydrophilicity / hydrophobicity, organic dyes can change the bioadhesive properties of various polymers when incorporated into a polymer matrix or bound to a polymer surface. Examples of dyes that affect biofouling properties include, but are not limited to: acidic fuchsin, alcian blue, alizarin red s, auramin o, azul a and b, bismarck brown y, brilliant cresyl Blue ald, brilliant green, carmine, cibacron blue 3GA, congo red, cresyl violet acetate, crystal violet, eosin b, eosin y, erythrocin b, fast green fcf, giemsa, hematylin, indigo carmine, janus green b, jenner dye , Oxalic acid malachite green, methyl blue, methylene blue, methyl green, methyl violet 2b, neutral red, nile blue a, orange II, orange G, orcein, para chloride Saniline, Phloxine b, Pyronin b and y, Reactive Blue 4 and 72, Reactive Brown 10, Reactive Green 5 and 19, Reactive Red 120, Reactive Yellow 2, 3, 13 and 86, Rose Bengal, Safranin o , Sudan III and IV, Sudan Black B and Toluidine Blue.

(Dispersant)
Preferred dispersing agents include hydrophilic polymers and wetting agents. The amount of dispersant in the formulation is less than about 80%, more preferably less than about 75% of the weight of the formulation. The most preferred polymer used as a dispersant is polyvinylpyrrolidone.

(II. Method for producing composition)
(A. Formation of nanoparticles)
In order to form drug nanoparticles, the drug is dissolved in a suitable solvent. The solution is then quickly diluted by adding a non-solvent solution. In general, the resulting nanoparticles are stable and do not irreversibly aggregate. This simplifies the recovery of the particles and the recovery is generally performed by methods such as centrifugation and filtration. The nanoparticles are then redispersed in a suitable solvent before use.

  The method used to form the nanoparticulate material can be easily scaled up as a batch or continuous method.

(solvent)
Suitable solvents have the property of dissolving the drug at a useful concentration, which concentration is at least about 0.5% (w / v), preferably at least about 2% (w / v), more preferably 5 More than 10% (w / v) or 10% (w / v). In general, the drug dissolves at a concentration well below its solubility limit. Furthermore, the solvent has as low a toxicity as possible and can be easily removed from the formed product by heat or vacuum. The solvent is completely or at least partially miscible with one or more non-solvents.

(Non-solvent)
Non-solvents are also selected for their low toxicity and easy removal. Important conditions for non-solvent selection are that the drug is not soluble in the non-solvent and that the non-solvent is sufficiently miscible with the solvent to form a single solvent phase after mixing. The solvent / non-solvent ratio is sufficient to make the mixed solution non-solvent for the drug. In order to prevent waste of the drug during processing, the solubility of the drug in the mixed solution is preferably low, for example 0.1% (w / v) or less. Preferably, the solvent and non-solvent are selected such that the absolute value of the difference in their solubility parameters is less than about 6 (cal / cm ³ ) ^1/2 . For example, when the solvent is an alcohol such as ethanol, the non-solvent is water or an aqueous solution. If the non-solvent is not water miscible, for example dichloromethane (methylene chloride), a suitable non-solvent is a non-polar solvent such as an alkane.

  Since the solvent and non-solvent are miscible, it is not necessary to stir to form the nanoparticles. In small volumes, the solvent and non-solvent can be mixed well by pouring one into the other. The drug / solvent solution may be poured into a volume of non-solvent, or the non-solvent may be poured into the drug / solvent solution. At large volumes, it may be convenient to stir one liquid while adding the other. Or, particularly in mass production, the solvent and non-solvent can be mixed continuously as an appropriate ratio flow. Agitation, if applied, need only be sufficient to prevent laminar flow of the streams.

  Because of their miscibility, the natural scale of liquid mixing is small. This is in contrast to mixing immiscible liquids, where surface tension tends to agglomerate the discontinuous phase and thus vigorous stirring is required to reduce particle size. Thus, when the two solutions are mixed, the drug precipitates as very fine particles, generally less than 5 microns in diameter.

  Any set of solvents and non-solvents in which the liquid is miscible and chemically and physically compatible with the drug may be used. “Chemical compatibility” means the absence of a chemical reaction between a solvent and a drug, excluding reversible changes such as ionization of acid groups in water. “Physical compatibility” means the absence of significant denaturation of macromolecular drugs such as proteins.

B. Formation of nanoparticles containing surfactants or other excipients and drugs
One or more surfactants or other excipients, such as bioadhesives, can be added to the drug in a number of ways. Surfactants may be applied at one or more of several stages in the nanoparticle production and dispersion process of the present invention. First, the surfactant may be present in the initial solution of the drug or other nanoparticle forming material. Second, it may be present in a non-solvent that is mixed with the drug solution that forms the nanoparticles.

  The third method involves adding a surfactant to the drug solution prior to precipitation with a non-solvent. This is the preferred method for small molecule surfactants.

  The fourth method involves dissolving the surfactant in the same solvent used to dissolve the drug. Next, the surfactant solution is mixed with a non-solvent. The non-solvent is preferably the same as the non-solvent mixed with the drug solution; if different, the non-solvent for the surfactant should also be non-solvent for the drug. The drug solution is separately mixed with a non-solvent. The two mixtures of solvent and non-solvent are then combined and drug nanoparticles are collected. This method is particularly suitable when the surfactant is a macromolecular surfactant or dispersant.

  The fifth method involves allowing some agglomeration of the particles during particle collection and then applying an appropriate dispersant as it is taken to use the particles. This method is particularly effective for medical and veterinary applications. As shown in the examples below, the addition of a suitable disaggregating surfactant can significantly increase the bioavailability of the nanoparticulate drug. The mechanism of this increase is believed to be the reversal or prevention of particle aggregation before or during ingestion or infusion.

(III. Use of composition)
Any medical or veterinary condition that can be treated with a drug can be treated using a nanoparticulate drug. In a preferred embodiment, the formulation is administered for the treatment of diseases such as cancer, oral vaccine administration, or any other medical or nutritional purpose that requires uptake from the mucosa of the drug or bioactive agent being transported. Is done.

  The drug nanoparticles can be administered to the patient by various routes. These routes include, but are not limited to: oral transport by absorption into the tissues of the oral cavity, gastrointestinal tract, and other parts of the body; transport to the nasal mucosa and lung (pulmonary); Transport to the skin, or transdermal transport; transport to other mucous membranes and epithelium including the reproductive and urinary tract (vagina, rectum, ureter); parenteral transport by circulation; and locally implanted Transport from depot or device.

(Example)
As shown in the examples, nanoparticulate drug formulations are used to enhance the transport of poorly soluble drugs through intestinal tissue, thereby allowing hydrophobic drugs to be administered orally rather than parenterally To do.

In the examples below, the material was obtained from an experimental material supplier and was a suitable brand for biomedical research. The substance / supplier combinations listed here were chosen for convenience:
Paclitaxel: Hauser Inc. Span 85 and Span 80: Sigma; PVP (polyvinylpyrrolidone), MW 40,000 (listed in the table), and pentane: EM Science; dichloromethane: Burdick &Jackson; TWEEN® 20: Malinckrodt; PEG (polyethylene glycol) ), MW4500 (described in the table): Spectrum Chemicals; EUDRAGIT (registered trademark) S100, MW135,000: Rohm &Hass; PLGA (polylactide-co-glycolide), 50:50, RG503 (without MW description): Boehringer Ingelheim; fumaric acid and sebacic acid: Aldrich.

Examples are formulations of taxane nanoparticles and / or microparticles such as paclitaxel, preferably biodegradable pharmaceuticals such as poly (lactide-co-glycolide) (“PLGA”). Encapsulated or dispersed in an acceptable polymer, most preferably further containing bioadhesion promoters such as FeO, Fe ₂ O ₃ , Fe ₃ O ₄ and fumaric anhydride oligomers, most preferably polyvinyl It shows that a formulation further containing a dispersant such as pyrrolidone ("PVP") has been developed. By inclusion in phase-inverted particles taken up by GI epithelial cells after oral administration, paclitaxel can be detected in blood and plasma by HPLC analysis. A bioavailability level of 5-15% is common. This is a nano- and microparticle formulation that delivers paclitaxel and / or other drugs with low water solubility and / or low absorption from the gastrointestinal tract after oral administration.

Formulations include polymers such as polylactic acid (PLA), poly-lactide-co-glycolide (PLGA) and poly (fumaric acid-co-sebacic anhydride), and FeO / Fe ₂ O ₃ , fumaric anhydride oligomers May contain polyvinylpyrrolidone and paclitaxel, or may be a combination of the above ingredients, including the formation of nano / micro particles of taxanes alone. In a preferred production process, all components except FeO / Fe ₂ O ₃ are dissolved in an organic solvent such as dichloromethane, acetone, chloroform, ethyl acetate and passed through a 0.2 μm PTFE filter. Next, FeO / Fe ₂ O ₃ is added and the resulting solution / suspension is bath sonicated for 2-5 minutes. Immediately place the solution / suspension in a pressure vessel containing a non-solvent such as pentane, hexane, heptane or petroleum ether present in a volume 15 to 100 times the volume of the organic solvent. The solution / suspension is self-dispersed or stirred if necessary to form solution / suspension nano / micro liquid particles. As the solvent exits the liquid particles and enters the non-solvent, nano / micro particles encapsulating the drug are rapidly and spontaneously formed. The particles are removed by filtration and vacuum dried to remove residual solvent and / or non-solvent.

  These taxane formulations were tested in vivo and were shown to yield 5-15% oral bioavailability when calculated for IV volumes that produced the same plasma AUC when observed for a given oral dose. .

(II. Formation of nano- or microparticles)
In a preferred embodiment, the formulation is in the form of nano- or microparticles and may be in the form of microcapsules, microspheres or microparticles. As described above, various polymers can be used to form microspheres, and the polymer surface of the microspheres is a metal compound that improves the bioadhesive properties of the microspheres, such as the ability of the microspheres to adhere to the mucosa. And / or oligomers are incorporated therein. Metal compounds and / or oligomers such as water insoluble metal oxides that improve the bioadhesive properties of the polymer are preferably incorporated into the polymer prior to the formation of the microspheres. As used herein, the term “microsphere” encompasses microparticles and microcapsules having a core of a material different from the outer wall. In general, microspheres have a diameter from nanometers to about 5 mm. For all methods, solvent evaporation, hot melt microencapsulation, solvent extraction, spray drying and phase inversion, the particle size is affected by the stirring speed, preferably less than 10 micrometers, more preferably less than 5 micrometers, More preferably, particle sizes smaller than 2 micrometers, more preferably smaller than 1 micrometer can be obtained. Microspheres may consist exclusively of bioadhesive polymers that incorporate metal compounds such as water-insoluble metal oxides, or outer coatings of bioadhesive polymers that incorporate metal compounds or other mucoadhesive agents. You may just have it.

  In one embodiment, polylactic acid microspheres can be produced using methods that include solvent evaporation, hot melt microencapsulation and spray drying. Polyanhydrides produced from bis-carboxyphenoxypropane and sebacic acid or poly (fumaric acid-co-sebacic acid) can be produced by hot melt microencapsulation. Polystyrene microspheres can be produced by solvent evaporation. Hydrogel microspheres such as alginate, chitosan, alginate / polyethyleneimine (PEI) and carboxymethylcellulose (CMC) are disclosed in PCT WO 93/21906 published November 11, 1993. The polymer solution can be made by dripping from a reservoir through a microfluidic particle forming device and into a stirred ion bath.

  In a preferred embodiment, the particles are nanoparticles formed by phase inversion as described in detail below.

(Solvent evaporation)
Methods for forming microspheres using the solvent evaporation method are described in E.C. Mathiowitz et al. Scanning Microscopy, 4: 329 (1990); R. Beck et al., Fertil. Steril. 31: 545 (1979); Benita et al. Pharm. Sci. 73: 1721 (1984). The polymer is dissolved in a volatile organic solvent such as dichloromethane. The substance to be incorporated is added to the solution and the mixture is suspended in an aqueous solution containing a surfactant such as poly (vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent has evaporated to obtain solid microspheres. Various sizes (1-1000 micrometers) and forms of microspheres can be obtained by this method. This method is effective for relatively stable polymers such as polyester and polystyrene. However, unstable polymers, such as polyanhydrides, can degrade during the manufacturing process due to the presence of water. For these polymers, some of the following methods performed in fully anhydrous organic solvents are more effective.

(Hot melt microencapsulation)
Microspheres can be formed from polymers such as polyesters and polyanhydrides using hot melt microencapsulation methods such as those described in Mathiowitz et al., Reactive Polymer, 6: 275 (1987). In this process, it is preferable to use a polymer having a molecular weight of 3 to 75,000 daltons. In this method, the polymer should first be melted and then incorporated to be screened to less than 50 micrometers or micronized to less than 10 micrometers, preferably less than 5 micrometers, preferably less than 1 micrometer. Mix with solid particles of material. The mixture is suspended in an immiscible solvent (eg silicone oil) and then heated to a temperature 5 ° C. above the melting point of the polymer with continuous stirring. Once the emulsion has stabilized, it is cooled until the polymer particles solidify. The resulting microspheres are washed by decantation with petroleum ether to obtain a free flowing powder. Microspheres with a size of 1-1000 micrometers are obtained by this method.

(Solvent extraction)
This method is designed primarily for polyanhydrides and is disclosed, for example, in PCT WO 93/21906 published November 11, 1993. In this method, the material to be incorporated is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent such as dichloromethane. This mixture is suspended in an organic oil, such as silicone oil, by stirring to form an emulsion. Microspheres of 1 to 300 micrometers can be obtained by this method.

(Spray drying)
A method for forming microspheres using a spray drying method is disclosed in US Pat. No. 6,262,034 to Mathiowitz et al. In this method, the polymer is dissolved in an organic solvent such as dichloromethane. A known amount of material to be incorporated is suspended (insoluble material) or co-dissolved (soluble material) in the polymer solution. Next, the solution or dispersion is spray-dried. Obtain 1-10 micrometer microspheres. This method is useful for producing microspheres for intestinal imaging. Using this method, in addition to the metal compound, a diagnostic imaging agent such as a gas can be incorporated into the microsphere.

(Phase inversion)
Phase inversion nanoencapsulation (PIN) is a method that involves the spontaneous formation of discrete microparticles. This one-step method does not require emulsification of the solvent phase in the non-solvent phase. Under appropriate conditions, the low viscosity polymer solution can be phase-inverted into fragmented spherical polymer particles when added to a suitable non-solvent. Phase inversion phenomena have been applied to produce macro and microporous polymer membranes, hollow fibers and nano and micro particles that are formed at low polymer concentrations. PINs are disclosed in US Pat. Nos. 6,143,211 and 6235224, Mathiowitz et al., Each incorporated herein by reference.

  During formation of PIN products using prior art PIN methods, significant aggregation of primary particles suspended in non-solvent can occur within 30 seconds of the initial injection of the polymer solution. The reason for aggregation is thought to be due to the interaction between the polymer and the non-solvent, or the interaction of the polymer itself. The interaction with the non-solvent is polymer dependent. An example is the interaction of PLGA based PIN particles with n-heptane. PIN particles consisting of 12K PLGA (50:50 L: G) aggregate within 30 seconds of injection, and similar particles based on 20:80 FA: SA polymer material show less agglomeration. This agglomeration of primary particles is very likely to cause an increase in the size of the final product when resuspended. This particle aggregation can affect the overall release of the drug or the ability of PIN particles to penetrate the mucosal epithelium.

  The method of the present invention maintains the primary particle size and also forms microparticles characterized by a homogeneous particle size to form a more accurate and reproducible transport system. Common microencapsulation methods produce a homogeneous particle size distribution of 10 μm to mm. Prior art methods attempt to adjust particle size by such parameters as stirring speed, temperature, polymer / suspension bath ratio, and the like. However, such parameters do not significantly narrow the particle size distribution. The PIN method, for example, can produce nanometer-sized particles that are relatively monodisperse in particle size. The improved PIN method of the present invention further reduces particle size by reducing particle agglomeration. By producing microparticles that are well defined and that have less variable particle size, the properties of the microparticles when used to release bioactive substances can be better tuned. Thus, the present invention allows improvements in the manufacture of sustained release formulations that are administered to patients.

  As disclosed in US Pat. No. 6,235,224, microspheres can be formed from a polymer using a phase inversion method, in which the polymer is dissolved in a good solvent and the drug Fine particles of the substance to be incorporated such as are mixed or dissolved in the polymer solution, and the mixture is poured into a strong non-solvent for the polymer to spontaneously produce polymer microspheres under suitable conditions. In the fair, the polymer covers the particles or the particles are dispersed in the polymer. Using this method, microparticles with a wide particle size range, for example, from about 100 nanometers to about 10 micrometers can be formed. Examples of polymers that can be used include polyvinylphenol and polylactic acid. Substances that can be incorporated include, for example, imaging agents such as fluorescent dyes, or biologically active molecules such as proteins or nucleic acids.

(Protein microencapsulation)
Protein microspheres can be formed by phase separation in a non-solvent followed by solvent removal, as disclosed in US Pat. No. 5,271,961 to Mathiowitz et al. Proteins that can be used include prolamins such as zein. In addition, mixtures of proteins or mixtures of proteins and bioerodible polymer materials such as polylactide can be used. In one embodiment, the prolamin solution and the substance to be incorporated are contacted with a second liquid that has limited miscibility with the proline solvent and the mixture is agitated to form a dispersion. The prolamin solvent is then removed to form stable prolamin microspheres without cross-linking or heat denaturation. Other prolamins that can be used include gliadin, hordein and kafilin. Substances that can be incorporated into the microspheres include pharmaceuticals, pesticides, nutrients and imaging agents in addition to metal compounds.

(Low temperature casting of microspheres)
A method for cryogenic casting of controlled release microspheres is disclosed in US Pat. No. 5,019,400 to Gombotz et al. In this method, the polymer is dissolved in a solvent along with the dissolved or dispersed material to be incorporated, and the mixture is atomized into a container containing a liquid non-solvent at a temperature below the freezing point of the polymer-material solution, thereby Freeze the liquid particles. As the liquid particles and the non-solvent for the polymer warm, the solvent in the liquid particles thaws and is extracted into the non-solvent, thereby curing the microspheres.

  In addition to metal compounds, biological materials such as proteins, short peptides, polysaccharides, nucleic acids, lipids, steroids and organic and inorganic drugs can be incorporated into the microspheres. Polymers that can be used to form the microspheres include, but are not limited to, poly (lactic acid), poly (lactic acid-co-glycolic acid), poly (caprolactone), polycarbonate, polyamide and polyanhydrides. Microspheres produced by this method are generally 5 to 1000 micrometers, preferably about 30 to 50 micrometers. However, smaller microspheres can be formed by using appropriate nozzles.

(Double-layer microcapsule)
A method for making multilayer polymer microspheres is disclosed in US Pat. No. 5,985,354 to Mathiowitz et al. In one embodiment, two hydrophilic polymers are dissolved in an aqueous solution. The substance to be incorporated is dispersed or dissolved in the polymer solution and the mixture is suspended in the continuous phase. The solvent is then slowly evaporated to form microspheres having an inner core formed by one polymer and an outer layer of a second polymer. The continuous phase is an organic oil, a volatile organic solvent, or an aqueous solution containing a third polymer that is not soluble in the first mixture of polymers and causes phase separation of the first two polyars when the mixture is stirred. It's okay.

  Multilayer polymeric drug, protein or cell delivery devices can be made from two or more hydrophilic polymers using this method. Any two or more different biodegradable or non-degradable water-soluble polymers may be used that are not mutually soluble at a specific concentration as defined by the phase diagram. Multilayer microcapsules have a uniformly sized polymer layer and can incorporate substances in addition to metal compounds, including drugs or cells, or diagnostic agents such as dyes.

  A microsphere containing a polymer core formed from a first polymer and a homogeneous coating of a second polymer and a material incorporated into at least one polymer is disclosed in U.S. Pat. No. 4,861,627. Can be manufactured.

(Hydrogel microspheres)
Microspheres formed from gel-type polymers such as alginate are produced by conventional ion gelation methods. The polymer is first dissolved in an aqueous solution and mixed with the substance to be incorporated, then extruded through a micro liquid particle forming device, and in some cases a stream of nitrogen gas is used to break off the liquid particles. A slowly stirred ion curing bath is placed below the extrusion apparatus to receive the formed micro liquid particles. The microspheres are incubated in the bath for 20-30 minutes to allow sufficient time for gelation to occur. The particle size of the microspheres is adjusted by using various sized extruders or by changing the flow rate of nitrogen gas or polymer solution.

  Chitosan microspheres can be made by dissolving the polymer in an acidic solution and crosslinking it with tripolyphosphate. Carboxymethylcellulose (CMC) microspheres can be made by dissolving the polymer in an acidic solution and precipitating the microspheres using lead ions. Alginate / polyethyleneimide (PEI) can be produced to reduce the amount of carboxyl groups on the alginate microcapsules. The advantage of these systems is the ability to use various chemistries to further modify their surface properties. In the case of negatively charged polymers (eg, alginate, CMC), positively charged ligands (eg, polylysine, polyethyleneimine) of various molecular weights can be ionically bound.

In a preferred embodiment, nano- and microparticle formulations for transporting paclitaxel and / or other drugs with low water solubility and / or low absorption from the gastrointestinal tract after oral administration use the following materials: Manufactured: polymers such as polylactic acid (PLA), polylactide-co-glycolide (PLGA) and poly (fumaric acid-co-sebacic anhydride) (poly (FA: SA)), and FeO / Fe ₂ O ₃ , fumaric anhydride oligomer, polyvinylpyrrolidone, and paclitaxel, or combinations of the above components (including the formation of nano / micro particles of paclitaxel only). These are preferably prepared containing accelerators such as metal oxides, bioadhesive oligomers, and dispersants such as PVP. All components except FeO / Fe ₂ O ₃ are dissolved in an organic solvent such as dichloromethane, acetone, chloroform, ethyl acetate (but not limited to) and passed through a 0.2 μm PTFE filter. Next, FeO / Fe ₂ O ₃ is added and the resulting solution / suspension is bath sonicated for 2-5 minutes. Alternatively, FeO / Fe ₂ O ₃ is added along with other ingredients and filtered or not filtered and the suspension is sonicated. Immediately place the solution / suspension in a pressure vessel containing a non-solvent, such as but not limited to pentane, hexane, heptane or petroleum ether, present in a volume 15 to 100 times the volume of the organic solvent. . The solution / suspension is self-dispersed or stirred if necessary to form solution / suspension nano / micro liquid particles. As the solvent exits the liquid particles and enters the non-solvent, nano / micro particles encapsulating the drug are rapidly and spontaneously formed. The particles are removed by filtration and dried in vacuo to remove residual solvent and / or non-solvent.

  This method combines a polymer, a dispersant and a taxane in an effective amount of solvent to form a continuous mixture and introduces the mixture into an effective amount of non-solvent to cause spontaneous formation of the nanoencapsulated product. May be performed. This method is a modification of the PIN method that incorporates the use of dispersants.

  The term “dispersant” encompasses “solvent soluble dispersants” as well as “solvent insoluble dispersants”, which can include water soluble and water insoluble materials, and can be micronized to obtain better final effectiveness. In some cases. As used herein, a “solvent soluble dispersant” is a solvent soluble material that is an organic solid at room temperature or is amphiphilic and prevents aggregation / fusion of PIN products during formation and collection. means. These compounds are added to and soluble in the polymer solution phase. Solvent soluble dispersants include, but are not limited to, natural and synthetic water soluble polymers or glidants such as polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), starch and lecithin.

PVP is a preferred solvent-soluble dispersant because it is soluble in the polymer solution phase and is also soluble in water and therefore precipitates when added to the non-solvent phase. PVP (C ₆ H ₉ NO) _n (also called povidone, polyvidone, poly [1- (2-oxo-1-pyrrolidinyl) ethylene]) is a synthetic polymer with a molecular weight of 2500-3,000,000. PVP has been used with solid dosage forms, where it acts as a non-toxic binder in tablets. PVP is also water soluble and is commonly used as a suspension stabilizer for many microparticle or microencapsulated formulations. It has been recognized as an excipient in most oral administrations because this compound is not absorbed through the intestine or mucosal surface and renders it non-toxic when consumed.

  PVP is added directly to the polymer solution prior to spontaneous particle formation. PVP can be added at a concentration of 0.1-50% of the total polymer content. Existing PIN methods allow for 0.1-20% (wt / vol) total polymer concentration in the solvent phase. PVP is not used in the PIN method to change the size of the primary polymer particles themselves. This particle size is determined by the operating parameters of the PIN method. Instead, the PVP additive prevents agglomeration of these primary particles into larger agglomerates, thereby producing an increased effective particle size. PVP can be used in the initial polymer solution to maintain the original primary particle size and prevent the general distribution of PIN material composed of particles and agglomerates. PVP can do this by incorporating it into the polymer particle matrix itself or by forming a coating around the primary polymer microparticles.

  Additional benefits are also obtained by using PVP in formulations using the PIN method. For low water-soluble drugs, PVP coatings can have the added advantage of changing the release characteristics of the substance by improving the solubility of the drug. PVP can be added to the PIN process and the PVP / PIN product can be tableted into a dosage form either directly or using additional additives. This dosage form can benefit from the binding properties of PVP itself and / or its action as a suspension enhancer upon reconstitution.

  Insoluble dispersants can also be used. The method is performed using PIN, but the insoluble dispersant is added to the non-solvent rather than the polymer solution. As used herein, “solvent insoluble dispersant” means an insoluble material that prevents aggregation / fusion of the PIN product during formation and collection. Solvent insoluble dispersants are organic or inorganic molecules of <100 micrometers, preferably <50 micrometers, most preferably <25 micrometers. These dispersants can be micronized to reduce their particle size prior to addition to the solvent or non-solvent. These substances, like PVP, may or may not dissolve during the reformation of the PIN product in water, but, like PVP, are pharmaceutically acceptable additives. They also act to reduce particle agglomeration during the PIN. The PIN method may be performed using a solvent soluble dispersant or a solvent insoluble dispersant or both.

  The dispersant can be added to the formulation using any of several methods. For example, the mixture of solvent, polymer, dispersant, and taxane-containing aqueous solution is frozen and then dried, preferably by vacuum, to remove water. After drying the frozen mixture, the dry mixture is redissolved in the solvent before being added to the non-solvent. In a preferred embodiment, the solvent, polymer, dispersant and mixture of materials are frozen in liquid nitrogen. Regardless of solubility, the dispersant can be micronized by two or more methods to obtain a smaller particle size, thereby increasing the effectiveness of the dispersant.

  In other embodiments, the dispersant is added to the non-solvent and solvent prior to introducing the solvent mixture into the non-solvent. In yet other embodiments, the dispersant is added only to the non-solvent prior to introducing the solvent mixture into the non-solvent. In still other embodiments, the dispersant is added to the solvent and non-solvent after introduction of the solvent mixture into the non-solvent, or the inhibitor is added only to the non-solvent after introduction of the solvent mixture into the non-solvent. In some embodiments, the dispersant concentration in the solvent is 0.01% to 10% (wt / vol) and in the non-solvent it is 0.1% to 20% (wt / vol).

  The solvent / non-solvent volume ratio is believed to be important in reducing particle aggregation or coalescence. The use range of the solvent / non-solvent volume ratio is 1:10 to 1: 1,000,000. In one embodiment, the solvent / non-solvent use range is from 1:10 to 1: 200.

  The resulting particles have an average particle size of 10 nanometers to 10 micrometers. In some embodiments, the particles have an average particle size of 10 nanometers to 5 micrometers. In yet other embodiments, the particles have an average particle size of 10 nanometers to 2 micrometers, or 10 nanometers to 1 micrometer.

(III. Administration of formulation)
The formulation is generally administered orally to patients in need thereof based on the disease to be treated or prevented and the known pharmacokinetics of the taxane. Administration may be via the lungs, nose, rectum or vagina. The drug may be administered once or more daily as needed. The drug particles may be administered as a specific formulation in capsules, auxiliaries or suspensions using materials and methods known to those skilled in the art.

  The invention will be further understood with reference to the following ratio-limiting examples.

Example 1: Production of bioadhesive nano and microparticulate taxane formulations
Paclitaxel (30% w / w), fumaric anhydride oligomer (10% w / w), PVP (2.8% w / w), and PLGA (45.4% w / w) in an amount of dichloromethane Dissolved and passed through a 0.2 micrometer PTFE filter. Then Fe ₃ O ₄ (11.8% w / w) was added and the whole mixture was bath sonicated for 2 minutes. This mixture was immediately dispersed in an amount of non-solvent, thereby obtaining a solvent volume / non-solvent volume ratio of 1: 100. The particles obtained from the phase inversion step were pressure filtered from solvent / non-solvent under nitrogen gas, collected and vacuum dried to remove residual solvent and / or non-solvent.

Example 2: Production of bioadhesive nano and microparticulate taxane formulations
A formulation was prepared as in Example 1 except that the drug content was increased to 50% (w / w) and the content of the other components was increased proportionally. The average relative bioavailability was 4.8% (+/− 1.6) (SEM).

Example 3: Production of bioadhesive nano and microparticulate taxane formulations
A formulation was prepared as in Example 1 except that the% content of PVP was tripled and the content of other components (except for the same drug content) was reduced proportionally. The average relative bioavailability was 7.5% (+/− 1.3) (SEM).

Example 4: Production of bioadhesive nano and microparticulate taxane formulations
Similar to Example 1 except that the% content of the fumaric anhydride oligomer was doubled and the content of other components (except for the same drug content as in Example 1) was reduced proportionally. The formulation was manufactured. The average relative bioavailability was 5.9% (+/− 0.6) (SEM). When the fumaric anhydride oligomer% was tripled, the content of other components (except for the same drug content as in Example 1) was reduced proportionally, with an average relative bioavailability of 7.8. % (+/− 1.1) (SEM).

Example 5: Production of bioadhesive nano and microparticulate taxane formulations using polyanhydride base polymer
Paclitaxel (30%, 50% and 70% w / w) and poly (fumaric acid-co-sebacic acid) (poly (FA: SA)) (20:80) are dissolved in dichloromethane and dissolved in 0.2 micrometer. The mixture was passed through a PTFE filter and bath sonicated for 2 minutes. This mixture was immediately dispersed in the non-solvent, thereby producing a solvent volume / non-solvent volume ratio of 1: 100. The particles obtained from the phase inversion step were pressure filtered from solvent / non-solvent under nitrogen gas, collected and vacuum dried to remove residual solvent and / or non-solvent.

Example 6: Production of nano and microparticulate taxane formulations
Paclitaxel was dissolved in dichloromethane to obtain a 3% (w / w) solution. The solution was passed through a 0.2 micrometer PTFE filter and bath sonicated for 2 minutes. This solution was immediately dispersed in the non-solvent, thereby yielding a solvent volume / non-solvent volume ratio of 1: 100. The particles obtained from the phase inversion step were pressure filtered from solvent / non-solvent under nitrogen gas, collected and vacuum dried to remove residual solvent and / or non-solvent.

Example 7: Bioavailability test of bioadhesive taxane formulation administered orally to rats
FIG. 1 is a graph comparing the average relative bioavailability of various oral formulations. Six oral formulations containing paclitaxel were tested. The columns in FIG. 1 are described below from left to right. The height of each column represents the average relative bioavailability after oral administration of 48 mg paclitaxel / kg rats. Column A shows the results of administration of a 30% paclitaxel / PLGA-PIN formulation containing a bioadhesive excipient as described in Example 1 (160 mg formulation / kg rat). Column B shows the results of administration of a 30% paclitaxel / PLGA-PIN formulation containing no bioadhesive excipient (160 mg formulation / kg rat). Column C shows the results of co-administration (160 mg formulation / kg rat) of a blank PLGA formulation with bioadhesive excipient and free paclitaxel. Column D shows the results of administration of free paclitaxel (48 mg formulation / kg rat) stirred in 0.5% SLS / PBS for dissolution. Column E shows the results of administration of paclitaxel (48 mg formulation / kg rat) micronized with PIN as described in Example 6. Column F shows the results of administration of a paclitaxel / PLGA-PIN formulation containing a bioadhesive excipient (160 mg formulation / kg rat). Except for paclitaxel (formulation E) micronized by phase inversion (PIN) method and formulation F resuspended in distilled water (dH ₂ O), all preparations were administered for administration at 0.5 Resuspended in% SLS / PBS. Excipients in Formulations A, C and F were fumaric anhydride oligomer, polyvinylpyrrolidone (PVP) and iron oxide (FeO, Fe ₂ O ₃ and / or Fe ₃ O ₄ ).

  An amount of each formulation was administered directly into the stomach of rats by oral gavage to give 48 mg paclitaxel / kg rats. Blood samples were collected at specific time points into test tubes supplemented with heparin and centrifuged. Plasma was removed and prepared for paclitaxel content HPLC analysis by liquid-liquid extraction with diethyl ether. The ether was evaporated and the sample was reconstituted in an acetonitrile: water HPLC mobile phase and injected directly into the HPLC. Plasma paclitaxel concentration was plotted at each time point.

  Since the low dose (<10 mg / kg) IV pharmacokinetics of paclitaxel is not linear, the bioavailability of an orally administered drug yields the same area under the plasma drug concentration time curve (AUC) as observed for a given oral dose Calculate for IV dose. To do this, IV pharmacokinetic studies were performed at several doses, the resulting AUC was determined, and an equation representing the dose / AUC relationship was fitted to the data. This allows the calculation of the IV dose corresponding to the observed oral AUC. The fractional bioavailability (BA) of the oral dose is the ratio of the corresponding IV dose / actual oral dose (IV dose / oral dose) of the oral formulation.

Formulation A produced an average relative bioavailability of 8.5%.
Formulation B produced an average relative bioavailability of 3.8%.
Formulation C produced an average relative bioavailability of 1.0%.
Formulation D did not produce mean relative bioavailability (0.0%).
Formulation E produced an average relative bioavailability of 2.4%.
Formulation F produced an average relative bioavailability of 8.5%.

The relative bioavailability of excipient-containing paclitaxel / PLGA made using PIN was 8.5% (see FIG. 1, columns A and F). This result is in sharp contrast to the bioavailability of paclitaxel / PLGA (column B) without excipients, which was 3.8%, or paclitaxel alone (column E), which was 2.4%. Thus, the presence of PLGA and excipients clearly increases the relative bioavailability of the drug. Furthermore, when paclitaxel / PLGA is dispersed in 0.5% SLS / PBS (column A) or dH ₂ O (column F), it is believed that there is no difference in relative bioavailability.

Example 8: Bioavailability test of bioadhesive taxane formulation administered orally to rats
A formulation containing paclitaxel and poly (FA: SA) (20:80) described in Example 5 was tested in vivo in rats. For administration, the formulation was resuspended in 0.5% SLS / PBS.

  Each resuspension formulation was then administered directly to the rat stomach by oral gavage. Blood samples were collected at specific time points (1 hour, 2 hours, 4 hours, 8 hours, 14 hours and 24 hours) into heparinized tubes and centrifuged. Plasma was removed and prepared for paclitaxel content HPLC analysis by liquid-liquid extraction with diethyl ether. The ether was evaporated and the sample was reconstituted in an acetonitrile: water HPLC mobile phase and injected directly into the HPLC. In FIG. 2, plasma paclitaxel concentration is plotted at each time point.

  FIG. 2 is a graph comparing plasma paclitaxel concentrations over time after oral administration of paclitaxel-PIN formulations (A), (B), (C) and free paclitaxel (D) described in Example 5. Formulation A contained 30% (w / w) paclitaxel / poly (FA: SA) PIN (48 mg paclitaxel / kg rat). Formulation B contained 50% (w / w) paclitaxel / poly (FA: SA) PIN (80 mg paclitaxel / kg rat). Formulation C contained 70% (w / w) paclitaxel / poly (FA: SA) PIN (112 mg paclitaxel / kg rat). Formulation D contained free paclitaxel administered orally at the same drug dose as Formulation A (48 mg / kg rat). Rats were administered 160 mg / kg rat formulations A, B, and C, and 48 mg / kg rat formulation D.

  Plasma paclitaxel concentrations peaked at 0-5 hours for all doses tested. As shown in FIG. 2, 50% drug concentration (Formulation B) is higher than both 30% (Formulation A) and 70% (Formulation C) drug state steady state plasma concentrations at 7-20 hours It is clear to give Paclitaxel was detected in plasma samples at all concentrations tested (formulations A, B and C) up to 24 hours after dosing. In contrast, no paclitaxel was detected in plasma samples after administration of free paclitaxel (Formulation D).

(Example 9: Production and sizing of particles)
Paclitaxel (3 mg) was dissolved in 1 mL of dichloromethane (methylene chloride) to give a 0.3% (w / v) solution. This solution was dispersed in 50 mL petroleum ether, non-solvent.

  The particles from the phase inversion process are recovered from the solvent / non-solvent mixture by pressure filtration through a 0.2 micron PTFE filter under nitrogen gas, collected from the filter, vacuum filtered, and residual solvent and non-solvent. The solvent was removed. Many particles were less than 0.2 microns in diameter, but the particles formed a filter cake that filled the membrane pores and were recovered in high yield.

(Example 10: Precipitation of taxol particles using pentane as a non-solvent)
Paclitaxel (30 mg) was dissolved in 1 mL of dichloromethane to prepare a 3% w / v solution. The solution was poured into 100 mL pentane, non-solvent. The particles were collected as described in Example 9.

(Example 11: Precipitation of taxol particles using water as a non-solvent)
Paclitaxel (30 mg) was dissolved in 1 mL of acetone and poured into 100 mL of water. The particles were collected by filtration as described in Example 9. Paclitaxel (30 mg) was dissolved in 1 mL of ethanol, poured into 100 mL of water, and the particles were collected by filtration. This indicates that the non-solvent may be more or less polar than the solvent, provided that the solvent and the non-solvent are substantially miscible.

(Example 12: Co-precipitation of taxol using a surfactant)
Paclitaxel (90.4 mg), polyvinylpyrrolidone (PVP) 5.0 mg, and PLGA (polylactide-co-glycolide) 5.1 mg were dissolved in 3.3 mL of dichloromethane. The solution was poured into 315 mL pentane, non-solvent. The particles were collected by filtration and dried in vacuo.

Example 13: Alternative collection method: surfactant in non-solvent
Paclitaxel (100.4 mg) was dissolved in 3.3 mL of dichloromethane and precipitated in 330 mL of pentane containing 1.65 g of Span 80 (registered trademark of polyoxyethylated fatty alcohol). The particles were collected by centrifugation, frozen in liquid nitrogen and vacuum dried.

(Example 14: Precipitation of particles using other surfactant)
Span 80 (16.7 g) in solvent (not present in non-solvent), EUDRAGIT® S-100 (polyacrylate) (33.3 mg), PVP (33.3 mg), or polyethylene glycol The experiment described in Example 13 was repeated using (PEG) (33.3 mg) with similar results in each case.

  In another experiment, 300 mg of paclitaxel was dissolved in 6 mL of ethanol and the solution was poured into 30 mL of water. Next, TWEEN® 20 (200 mg) was added to the particle suspension. The particles were collected by filtration and dried in vacuo.

(Example 15: Scale-up of paclitaxel particle formation)
2400 mg of paclitaxel was dissolved in 80 mL of dichloromethane and poured into 8000 mL of pentane. The particles were collected by filtration and dried in vacuo.

  The numerical values used in these experiments (3%, 1: 100) were chosen for convenience and maintained for comparison. It is believed that the drug concentration in the solvent and its amount in the non-solvent can each be significantly reduced.

(Example 16: Addition of tissue adhesive to particles)
U.S. Pat. No. 4,891,225 discloses a copolymer of fumaric anhydride and sebacic anhydride prepared in house by conversion of acid to anhydride and polymerization of anhydride in toluene ( pFA: SA) (20:80) is thought to promote particle adhesion to the intestinal and other mucosa. Paclitaxel (2280 mg) and pFA: SA (120 mg) were dissolved in 80 mL of dichloromethane. The solution was poured into 8000 mL of pentane containing Span 85 (16 mL) used as a surfactant. The particles were collected by vacuum and dried in vacuo.

Example 17: In vivo test of bioavailability of oral paclitaxel formulation
The bioavailability of five formulations of orally administered paclitaxel was compared to conventional administration of an intravenous solution containing paclitaxel, ethanol and polyethoxylated castor oil. A sufficient amount of each formulation to deliver 48 mg paclitaxel / kg rats was administered directly into the rat stomach by oral gavage. Blood samples were collected at specific time points (0.5, 2, 4, 8, 24, 48, and 72 hours) into heparinized tubes and centrifuged. Plasma was removed and prepared for high performance liquid chromatography (HPLC) analysis of paclitaxel content by liquid-liquid extraction with diethyl ether. The ether was evaporated and the sample was reconstituted in an acetonitrile: water (70:30) HPLC mobile phase and injected directly into the HPLC machine. Plasma paclitaxel concentration was plotted at each time point of sampling.

  Since the low dose (<10 mg / kg) intravenous (IV) pharmacokinetics of paclitaxel is not linear, the bioavailability of the orally administered drug is the same as the area under the plasma drug concentration time curve observed for a given oral dose ( Calculate for the IV dose that yields (AUC). To do this, IV pharmacokinetic studies were performed at several doses, the resulting AUC was determined, and an equation representing the dose / AUC relationship was fitted to the data. This allows the calculation of the IV dose corresponding to the observed oral AUC. Oral dose fractional bioavailability (BA) is the ratio of the corresponding IV dose of the oral formulation / actual oral dose (IV dose / oral dose). The results of this bioavailability test are shown in FIG.

(5 formulations and their corresponding relative bioavailability)
Paclitaxel microspheres were prepared essentially as described in Example 2. In each case, the same amount (48 mg / kg) of paclitaxel was administered. In the leftmost column (a), microspheres were added to isotonic phosphate buffered saline (PBS) containing 0.5% sodium lauryl sulfate (SLS) and 0.1% PVP (polyvinylpyrrolidone; MW 40,000 D). I took. The relative bioavailability (BA) was 9.6%.

  In the second column (b), changes were made to Example 10 to form microspheres. PVP was not present in the paclitaxel solution. Instead, a separation solution (1% w / v) containing PVP dissolved in dichloromethane was precipitated in pentane. Next, PVP in pentane was immediately added to the drug particle suspension, the mixing ratio being 1 mg PVP (1% diluted with 100 volumes of pentane) for 11.25 mg paclitaxel (3% solution diluted with 100 volumes of pentane). Solution), ie, about 1.75 volumes of PVP for each 3.75 volume of paclitaxel (this gives the same PVP / paclitaxel ratio as Example 9). After mixing the granular paclitaxel and PVP solution, the nanoparticles were collected by filtration. Samples were taken in 0.5% SLS in PBS without adding additional PVP. When given to rats, the observed BA was 10.2%.

  In the middle column (c), paclitaxel nanospheres were formed as described in Example 2 and taken up in 0.5% SLS in PBS. No additional surfactant was added. The observed BA was only 2.4%. This low BA is believed to be due to inadequate resuspension of the nanoparticles in the absence of a suitable surfactant.

  In the fourth (blank) column (d), rats were given stored (as-purchased) paclitaxel soaked in 0.5% SLS in PBS for 36 hours. The observed BA was 0% (no paclitaxel was detected in the serum).

  In the rightmost column (e), stored paclitaxel was taken up in 0.5% SLS in PBS and administered immediately. 0.7% BA was observed.

  These results are shown graphically in FIG.

Example 18: Effect of sonication time, stability (time after sonication) and solvent / non-solvent ratio on particle size
Effect of time after particle resuspension following a single 3 minute bath sonication on the particle size of paclitaxel particles produced by PIN. All particles were made from a 3% paclitaxel solution in methylene chloride using 100 volumes of pentane at room temperature. The particles were collected by filtration and dried in vacuo. The particles were resuspended in 1.0% (w / v) PVP / 0.5% (w / v) sodium lauryl sulfate (“SLS”) / phosphate buffered saline (“PBS”). The result is shown in FIG. This experiment shows the stability of particle size (absence of reaggregation) after 3 minutes of bath sonication (note that the particle size scale on the left vertical axis has changed). The results show that the particles are stable for at least 60 minutes after sonication.

  Effect of bath sonication time on the size of paclitaxel particles prepared by PIN. Samples were resuspended and bath sonicated for a predetermined time and then measured immediately. The sized resuspension medium was 1.0% (w / v) PVP / 0.5% (w / v) SLS / PBS. The result is shown in FIG. The results show that in the conditions used, sonication times longer than 1 minute are necessary to obtain nanometer diameters. A standard sonication time of 3 minutes was selected. Note that at 2 minutes, under these conditions, the effective particle size decreased to a submicron value centered around 0.1 microns.

  Using the conditions established by these experiments, paclitaxel was used as a model drug to examine the effect of drug concentration and dilution rate in non-solvent. Control conditions in all experiments were 3% drug concentration, 1: 100 dilution in precipitation, and 3 minutes bath sonication after resuspension.

  Drug concentration affects particle size. The paclitaxel concentration in methylene chloride was varied. The precipitation ratio was 1 volume of methylene chloride / 100 volumes of pentane. The particles were collected, dried, resuspended and sonicated for 3 minutes. The effect of paclitaxel solution concentration (w / v) in dichloromethane (DCM) (before PIN processing) on the particle size of paclitaxel particles produced by PIN was measured. Samples were resuspended and then measured immediately after a single 3 minute bath sonication. The results are shown in FIG. 5c and indicate that lower DCM concentrations (less than 7%) result in smaller particle sizes (nanometer range).

  Effective particle size increases with increasing drug loading. The increase was modest in 1% -5% drug. The particle size distribution shows a gradual shift from 100 nm particles to about 500 nm diameter particles. At 7% and 9%, significant aggregation occurred, as judged by the appearance of 5-10 micron particles in the particle size distribution plot.

  The effect of the solvent / non-solvent ratio (during the PIN process) on the particle size of paclitaxel particles produced by PIN was measured. The solvent was dichloromethane and the non-solvent was pentane. Samples were resuspended and then measured immediately after a single 3 minute bath sonication. The sized resuspension medium was 1.0% (w / v) PVP / 0.5% (w / v) SLS / PBS. FIG. 5d shows the results for a solvent dichloromethane / non-solvent pentane ratio of 1: 100 to 1:05. All ratios yielded nanoparticles. Examples of dilution ratios are 1: 100 (standard), 1:50, 1:25, 1:10 and 1: 5. The 1: 100 and 1:50 dilutions are essentially the same; 1:25 dilutions show some increase in particles in the 0.3-0.5 micron range; particle size modes are 1:10 and 1 : Shift to 0.5 micron range at 5. The particle size distribution plot shows that this increase was not accompanied by a significant extension of the particle size to the multi-micron particle size range. The effect of additional sonication was not investigated. A dilution ratio of 1: 1 was also tested. The drug precipitates as a macroscopic precipitate with little evidence of fine particle formation; in one experiment, the precipitate spontaneously redissolves, which is a 1: 1 ratio, while paclitaxel is soluble in it. It means very close to the limiting concentration of pentane in some dichloromethane. Temperature can also be a variable in solubility.

  This data set shows that a concentration of 1% at a dilution factor of 50 is nearly optimal to obtain fine particles in the 100 nm range using paclitaxel in dichloromethane precipitated with pentane. For 500 nm particles that are stable to light sonication, a 5% drug concentration and a 1: 5 dilution ratio are nearly optimal in terms of minimizing solvent use.

  In other experiments, 3% of the drug carbamazepine in dichloromethane is precipitated in 100 volumes of pentane. According to electron microscopy, the particles were mainly acicular, about 1 micron in diameter and 50 microns in length. Some 1 micron spherical particles were also observed. In a similar experiment using the drug itraconazole, electron microscopy yielded approximately spherical particles of about 0.1-0.5 microns in diameter.

  These experiments require specific conditions for the reliable formation of nanoparticles, which is not necessarily a consequence of dissolving the drug in a solvent and precipitating it in a non-solvent. It shows that. It further shows that once information about the general range from which nanoparticles can be obtained is obtained, optimizing the conditions for a particular drug requires only a reasonable amount of experimentation.

(Example 19: Effect of paclitaxel nanoparticles orally administered to mice on tumor growth)
Paclitaxel nanoparticles prepared as described above (without using bioadhesive or surfactant) were orally administered to female nude mice inoculated with breast tumor cells twice daily for 5 days. Nanoparticles were resuspended at 11.25 mg / mL in 0.1% PVP / 0.5% SLS / PBS and administered by oral gavage to give theoretical doses of 24, 48 and 72 mg / kg. . The control was taxol in cremaphor administered by intravenous injection (30 mg / kg).

  The results are shown in FIG. The results show that taxol nanoparticles had about 17% bioavailability compared to taxol in cremaphor, resulting in some reduction in tumor growth, about 50 at a dose of 72 mg / kg. % Tumor volume reduction. Variables that can be used to increase effectiveness include higher drug loading, bioadhesive incorporation, and particle size optimization (less than 500 nm) to increase uptake.

FIG. 1 is a graph of particle size (μm) versus percent (number and amount) of paclitaxel particles formed by the methods disclosed herein. FIG. 2 is a bar graph comparing the relative bioavailability (± standard error) of paclitaxel for five oral formulations. FIG. 3 is a graph of the average relative bioavailability of paclitaxel phase-inversion nanoencapsulation (PIN) formulations and stored paclitaxel. FIG. 4 is a graph comparing paclitaxel plasma concentrations over time after oral administration of paclitaxel-poly (FA: SA) PIN formulations (A), (B), (C) and free paclitaxel. 5a-5b are graphs showing the effect of time after sonication on particle size (FIG. 5a); the effect of sonication time on particle size (FIG. 5b). Figures 5c-5d are graphs showing the effect of drug solution concentration on particle size (Figure 5c); and the effect of solvent / non-solvent ratio on particle size (Figure 5d). FIG. 6 is a graph of tumor size after administration of three doses of paclitaxel nanoparticles compared to injection of taxol in cremaphor.

Claims (31)

A method for preparing nanoparticles of therapeutic, prophylactic or diagnostic substances having a water solubility of less than about 0.1 w / v% at room temperature, comprising:
Dissolving a substance in a solvent to form a first solution;
Providing a non-solvent for the substance that is miscible with the solvent, and mixing the first solution and the non-solvent to form nanoparticles of a therapeutic, prophylactic or diagnostic substance. The nanoparticles form a population with at least 95% having a diameter of less than 1 micron;
Including the method. The method of claim 1, further comprising adding a surfactant or excipient. The method of claim 2, wherein the surfactant or excipient is added to the solvent. The method of claim 2, wherein the surfactant or excipient is added to the non-solvent. The method of claim 2, wherein the surfactant or excipient is added to the nanoparticles after the nanoparticles are formed. The method of claim 1, wherein the substance is selected from the group consisting of small molecule drugs, proteins, lipids, polysaccharides, proteoglycans and polynucleotides. The method of claim 1, wherein the substance is sufficiently hydrophobic to be insoluble in water. The method of claim 1, further comprising collecting the nanoparticles by centrifugation, filtration, lyophilization or spray drying. The method of claim 1, wherein less than about 1% of the nanoparticles have a diameter greater than about 1 micron. A population comprising at least 95% nanoparticles of a therapeutic, diagnostic or prophylactic agent having a diameter of less than 1 micron, wherein the material has a water solubility of less than about 0.1 w / v% at room temperature. 11. The population of claim 10, wherein the substance is selected from the group consisting of small molecule drugs, proteins, lipids, polysaccharides, proteoglycans and polynucleotides. 11. The population of claim 10, wherein the substance is sufficiently hydrophobic because it is insoluble in water. The population of claim 10, wherein at least 99% of the nanoparticles have a diameter of less than 1 micron. The population of claim 10, further comprising a bioadhesion promoter. The population of claim 10 further comprising a dispersant. The population of claim 10 further comprising a polymer. 11. The population of claim 10, comprising a polymer encapsulant having a bioadhesive bonded to or dispersed in the polymer. 18. The population of claim 17, wherein the bioadhesive is selected from the group consisting of bioadhesive metal compounds and bioadhesive organic molecules. The population of claim 10, wherein the nanoparticles are:
Dissolving the substance in a solvent to form a first solution;
Providing a non-solvent for the material, the non-solvent being miscible with the solvent; and mixing the first solution with the non-solvent to form nanoparticles;
A population formed by a method comprising: Nanoparticle or microparticle formulation for oral administration of taxanes that provides bioavailability of at least 5% of the bioavailability of taxanes when administered intravenously. 21. The formulation of claim 20, wherein the taxane is paclitaxel. 21. The formulation of claim 20, wherein the taxane is docetaxel. 21. The formulation of claim 20, wherein 90% of the volume or number of nanoparticles and microparticles has a diameter of less than 5 microns. 21. The formulation of claim 20, wherein 90% of the volume or number of nanoparticles and microparticles has a diameter of less than 1 micron. 21. The formulation of claim 20, wherein the taxane is present in a drug volume up to 70% by weight. 21. The formulation of claim 20, wherein the taxane is present at a drug volume of about 30-70% by weight. 21. The formulation of claim 20, further comprising a surfactant or excipient. 21. A method for treating a patient comprising administering to the patient a nanoparticle population of claim 10 or a formulation containing the nanoparticle formulation of claim 20. 30. The method of claim 28, wherein the formulation is selected from the group consisting of oral formulations, aerosols, topical formulations, parenteral formulations and implantable compositions. 30. The method of claim 28, wherein the formulation is administered orally. 30. The method of claim 28, wherein the formulation is administered to the pulmonary system.

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